Fluorescence Made Easier: Fluorescence Techniques for the Novice Episode 3:. Avoiding Those Pesky Artifacts: Sources of Error in Spectra and Steady State Measurements

Abstract

For those contemplating employing fluorescence methods in their experiments, there are some potential artifacts and sources of error that can invalidate results. With a little care, these artifacts can be avoided to produce the kind of results that will make the reviewers jump for joy (and accept your paper). We will cover some sources of error arising from issues arising from the sample itself, and basic problems with fluorometers. At the end we have a handy checklist for when your spectrum comes out looking really messed up.

Handy Checklist for Figuring Out Why Your Spectrum Looks Weird

You have your sample in your fluorometer and have figured out the wavelength to excite the fluorophore and the emission wavelengths you should scan, but instead of obtaining the nice Gaussian-shaped curve you expect, you get something insane. Here are some problems that might happen and how to fix them (which will save you time reading the text).

• 1. I don’t have a signal!

• Make sure your instrument is on and light is reaching the cuvette (this can be done by moving the monochromator to 500 nm, opening the top of the sample compartment and checking to see a beam of green light with a business card. If you don’t see light, make sure everything is ON, shutters are open, etc.

  • If you see a nice green beam going through your cuvette, then check to make sure that you are using the appropriate wavelengths for your scan and that you have enough probe.

    • If neither of these work, then you might have an unforeseen quencher in your cuvette. Try a different solvent, and/or a different fluorophore.

  If you see the green light hitting the front of the cuvette, but not going through the sample (like Fig. 11.1), then reduce the concentration of your probe by at least a factor of 10 and look again. If the beam gets weaker as it goes through the cuvette, you still need to dilute.

• 2. My spectrum starts high and crashes down, like an intense ski slope

  You probably have too much Rayleigh scattered light (Fig. 11.5). Start the scan at a higher (i.e. 5–10 nm) wavelength. Or use a cut-on optical filter to eliminate lower wavelength emission.

  Take a background spectrum to check for impurities – this should always be done even if your spectrum doesn’t look weird.

  Make sure there isn’t lint, debris or other stuff floating around in your cuvette.

  Also, make sure there aren’t tiny little bubbles in the cuvette due to pipetting cold solution directly into the room temperature cuvette.

• 3. The intensity of my spectrum is reduced every time I scan.

  Thermal heating from shining light on your sample may reduce the intensity. If your sample holder does not have a thermal jacket, then try to make sure your sample remains at the desired temperature through the scan. This might involve scanning quickly for cooler samples, or thermally equilibrating the sample in the optical module.

  Photobleaching, the light-induced breakdown of a fluorophore (usually by oxidation), can kill your fluorescence. Minimize how much you illuminate the sample with excitation as much as you can. Photobleaching can sometimes be avoided by purging the sample and the optical module with an inert gas such as Argon or Nitrogen. If your sample contains cells that require oxygen, you may need to use a different fluorophore if you can.

  Some samples (like cell suspensions) may settle during the time of the measurements, so that the cells are no longer in the excitation beam. Settling can be avoided by gently stirring the sample (if possible) or by increasing the density of the solvent to keep the sample buoyant.

• 4. My spectra has funny bumps.

  Take a background spectrum to make sure these aren’t due to solvent scattering and /or impurities. If not, then it is possible that the fluorophore itself has multiple peaks due to changes in ionization in the excited state , or a rich vibrational structure.